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My accidental encounter with protein hydrogen exchange (HX) at its very beginning and its continued development through my scientific career have led us to a series of advances in HX measurement, interpretation, and cutting edge biophysical applications. After some thoughts about how life brought me there, I take the opportunity to reflect on our early studies of allosteric structure and energy change in hemoglobin, the still-current protein folding problem, and our most recent forward-looking studies on protein machines.

Introduction: Patients (pts) with CLL may be at particular risk of severe COVID-19 given advanced age and immune dysregulation. Two large series with limited follow-up have reported outcomes for pts with CLL and COVID-19 (Scarfò, et al. Leukemia 2020; Mato, et al. Blood 2020). To provide maximal clarity on outcomes for pts with CLL and COVID-19, we partnered in a worldwide effort to describe the clinical experience and validate predictors of survival, including potential treatment effects. Methods: This international collaboration represents a partnership between investigators at 141 centers. Data are presented in two cohorts. Cohort 1 (Co1) includes pts captured through efforts by European Research Initiative on CLL (ERIC), Italian CAMPUS CLL Program, and Grupo Español de Leucemia Linfática Crónica. The validation cohort, Cohort 2 (Co2), includes pts from US (66%), UK (23%), EU (7%), and other countries (4%). There is no overlap in cases between cohorts. CLL pts were included if COVID-19 was diagnosed by PCR detection of SARS-CoV-2 and they required inpatient hospitalization. Data were collected retrospectively 2/2020 - 5/2020 using standardized case report forms. Baseline characteristics, preexisting comorbidities (including cumulative illness rating scale (CIRS) score ≥6 vs. The primary endpoint of this study was to estimate the case fatality rate (CFR), defined as the proportion of pts who died among all pts hospitalized with COVID-19. Chi-squared test was used to compare frequencies; univariable and multivariable analyses utilized Cox regression. Predictors of inferior OS in both Co1 and Co2 were included in multivariable analyses. Kaplan-Meier method was used to estimate overall survival (OS) from time of COVID-19 diagnosis (dx). Results: 411 hospitalized, COVID-19 positive CLL pts were analyzed (Co1 n=281, Co2 n=130). Table 1 describes baseline characteristics. At COVID-19 dx, median age was 72 in Co1 (range 37-94) and 68 in Co2 (range 41-98); 31% (Co1) and 45% (Co2) had CIRS ≥6. In Co1, 48% were treatment-naïve and 26% were receiving CLL-directed therapy at COVID-19 dx (66% BTKi ± anti-CD20, 19% Venetoclax ± anti-CD20, 9.6% chemo/chemoimmunotherapy (CIT), 1.4% PI3Ki, 4% other). In Co2, 36% were never treated and 49% were receiving CLL-directed therapy (65% BTKi ± anti-CD20, 19% Venetoclax ± anti-CD20, 9.4% multi-novel agent combinations, 1.6% CIT, 1.6% PI3Ki, 1.6% anti-CD20 monotherapy, 1.6% other). Most pts receiving CLL-directed therapy had it held at COVID-19 diagnosis (93% in Co1 and 81% in Co2). Frequency of most COVID-19 symptoms/laboratory abnormalities were similar in the two cohorts including fever (88% in both), lymphocytosis (ALC ≥30 x 109/L; 27% vs. 21%), and lymphocytopenia (ALC < 1.0 x 109/L; 18% vs. 28%), while others varied between Co1 and Co2 (p Median follow-up was 24 days (range 2-86) in Co1 and 17 days (1-43) in Co2. CFRs were similar in Co1 and Co2, 30% and 34% (p=0.45). 54% and 43% were discharged while 16% and 23% remained admitted at last follow-up in Co1 and Co2, respectively. The proportion of pts requiring supplemental oxygen was similar (89% vs. 92%) while rate of ICU admission was higher in Co2 (20% vs. 48%, p Conclusions : In the largest cancer dx-specific cohort reported, pts with CLL hospitalized for COVID-19 had a CFR of 30-34%. Advanced patient age at COVID-19 diagnosis was an independent predictor of OS in two large cohorts. This CFR will serve as a benchmark for mortality for future outcomes studies, including therapeutic interventions for COVID-19 in this population. The effect of CLL treatment on OS was inconsistent across cohorts; COVID-19 may be severe regardless of treatment status. While there were no significant differences in distribution of current lines of therapy between cohorts, prior chemo exposure was more common in Co1 vs. Co2, which may account for difference in OS. Extended follow-up will be presented. Disclosures Roeker: American Society of Hematology: Research Funding; Abbott Laboratories: Other: spouse with minority ownership interest ; AbbVie: Other: spouse with minority ownership interest . Scarfo:Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AstraZeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees. Abrisqueta:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau. Eyre:AbbVie: Consultancy, Honoraria, Other: travel support; Gilead: Consultancy, Honoraria, Other: travel support; Janssen: Consultancy, Honoraria, Other: travel support; KITE, AZ, Loxo Oncology at Lilly: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Muntañola Prat:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; participated in advisory boards; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; participated in advisory boards; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel grants; participated in advisory boards. Villacampa:AstraZeneca: Other: advisory role; Merck Sharp & Dohme: Honoraria. Leslie:AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; ADC therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; BeiGene: Speakers Bureau; KitePharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene/BMS: Speakers Bureau; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics/Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Epizyme: Speakers Bureau; Karyopharm: Speakers Bureau; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Allan:Acerta, Genentech, Abbvie, Sunesis, Ascentage, Pharmacyclics, Janssen, AstraZeneca, BeiGene: Consultancy; Celgene, Genentech, Janssen, TG Therapeutics: Research Funding; Abbvie, Janssen, AstraZeneca, Pharmacyclics: Honoraria. Furman:Incyte: Consultancy; Genentech: Consultancy; Sunesis: Consultancy; Pharmacyclics: Consultancy; Loxo Oncology: Consultancy; Oncotarget: Consultancy; Janssen: Consultancy, Speakers Bureau; TG Therapeutics: Consultancy, Research Funding; Abbvie: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy, Research Funding; Acerta: Consultancy; Verastem: Consultancy. Pagel:BeiGene, Astrazeneca, Loxo Oncology, Gilead: Consultancy. Hernandez-Rivas:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Rovi: Membership on an entity's Board of Directors or advisory committees. Patel:Genentech: Consultancy, Speakers Bureau; Adaptive Biotechnologies: Consultancy; Janssen: Consultancy, Speakers Bureau; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; BeiGene: Consultancy; Kite: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau; AstraZeneca: Consultancy, Research Funding, Speakers Bureau. Motta:Roche: Honoraria; Janssen: Honoraria. Lamanna:AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Verastem: Research Funding; Bei-Gene: Research Funding; TG Therapeutics: Research Funding; Acerta: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Vitale:Janssen: Honoraria. Kamdar:Roche: Research Funding. Österborg:BeiGene: Research Funding; Kancera: Current equity holder in publicly-traded company, Research Funding; Sanofi: Consultancy; Karolinska Univeristy Hospital, Stockholm, Sweden: Current Employment. Hanson:Janssen-Cilag: Research Funding; Gilead: Research Funding; AbbVie: Honoraria. Eichhorst:ArQule: Consultancy, Honoraria, Other: travel support, Research Funding; BeiGene: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: travel support, Research Funding; Oxford Biomedica: Consultancy, Honoraria, Other: travel support, Research Funding; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding; Novartis: Consultancy, Honoraria, Other: travel support, Research Funding. Reda:Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees. Varettoni:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel/accommodations/expenses; AbbVie: Other: Travel/accommodations/expenses; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees. Marchetti:Gilead: Consultancy; Novartis: Speakers Bureau; Amgen: Speakers Bureau; AbbVie: Other: Sponsored meetings; Takeda: Other: Sponsored meetings; Pfeizer: Other: Sponsored meetings. Munir:F. Hoffmann-La Roche: Consultancy, Other: Medical writing support, furnished by Scott Battle, PhD, of Health Interactions, was funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland; Alexion: Honoraria. Zabalza:Janssen: Honoraria, Other: travel grants; Roche: Other: travel grants; Novartis: Other: travel grants. Janssens:Amgen: Consultancy, Other: travel grants; speaker fees; Abbvie: Consultancy, Other: travel grants; speaker fees; Celgene: Consultancy, Other: travel grants; speaker fees; Janssen: Consultancy, Other: travel grants; speaker fees; Gilead: Consultancy, Other: travel grants; speaker fees; Novartis: Consultancy, Other: travel grants; speaker fees; Sanofi-Genzyme: Consultancy, Other: travel grants; speaker fees; Roche: Consultancy, Other: travel grants; speaker fees. Niemann:AstraZeneca: Honoraria, Research Funding; CSL Behring: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Danish Cancer Society: Honoraria, Research Funding; Novo Nordisk Foundation: Honoraria, Research Funding. Perini:Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau; Abbvie: Speakers Bureau. Patten:AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria. Marasca:Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Honoraria. Iyengar:Janssen: Honoraria; Gilead: Honoraria. Ferrari:Abbvie: Honoraria. El-Sharkawi:Roche: Other: Conference fees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Itchaki:Abbvie Inc: Consultancy, Research Funding. Ma:Novartis: Research Funding; Juno: Research Funding; Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding, Speakers Bureau; Kite: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Bioverativ: Consultancy, Honoraria; BeiGene: Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding; TG Therapeutics: Research Funding. Van Der Spek:AMGEN: Other: Teaching activities. Seymour:Seattle Genetics: Research Funding; Merck: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen/Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Genentech: Research Funding; Bristol-Myers Squibb: Research Funding. Rigolin:Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Mauro:Roche: Other; Octopharma: Other; Takeda-Shire: Other; Gilead: Other; Janssen: Other; Abbvie: Other. Laurenti:Janssen: Honoraria; Gilead: Honoraria; AbbVie: Honoraria; Roche: Honoraria. Levin:Janssen: Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Roche: Membership on an entity's Board of Directors or advisory committees, Other: travel compensation. Špaček:Gilead: Honoraria; Abbvie: Honoraria; Janssen: Honoraria. Walewska:AbbVie: Other: sponsored for educational meetings, Speakers Bureau; Janssen: Other: sponsored for educational meetings, Speakers Bureau; Gilead: Speakers Bureau; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Wiestner:Pharmacyclics LLC, an AbbVie Company; Acerta, Merck, Nurix, Verastem, and Genmab: Research Funding; National Institutes of Health: Patents & Royalties: and other intellectual property. Broom:Gilead: Other: Travel support, Speakers Bureau. Kater:Abbvie: Research Funding; Roche: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Genentech: Research Funding. Ujjani:AstraZeneca: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Verastem Oncology: Consultancy, Honoraria; Gilead/Kite: Consultancy, Research Funding; Atara: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; MorphoSys: Consultancy. Vandenberghe:Celgene: Other: sponsorship to attend Lugano lymphoma meeting in 2019; Gilead: Other: travel grants, Research Funding; Abbvie: Other: travel grants, Research Funding; Janssen: Other: travel grants; Roche: Other: travel grants, Research Funding. Chong:Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; KITE Pharma: Membership on an entity's Board of Directors or advisory committees. Pu:Takeda Pharmaceuticals: Consultancy. Brown:Janssen, Teva: Speakers Bureau; Gilead, Loxo, Sun, Verastem: Research Funding; Abbvie, Acerta, AstraZeneca, Beigene, Invectys, Juno/Celgene, Kite, Morphosys, Novartis, Octapharma, Pharmacyclics, Sunesis, TG Therapeutics, Verastem: Consultancy. Trentin:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria. Farina:Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Sanchez:Abbvie: Other: travel grants; Amgem: Other: travel grants; Janssen: Other: travel grants; Celgene: Other: travel grants; Roche: Other: travel grants. Shadman:Abbvie, Genentech, Astra Zeneca, Sound Biologics , Pharmacyclics, Verastem, ADC therapeutics, Beigene, Cellectar, BMS, Morphosys and Atara Biotherapeutics: Consultancy; Mustang Bio, Celgene, Pharmacyclics, Gilead, Genentech, Abbvie, TG therapeutics, Beigene, Astra Zeneca, Sunesis, Beigene: Research Funding. Foglietta:Janssen: Honoraria; Gilead: Honoraria. Jaksic:Roche: Honoraria; Janssen: Honoraria; Abbvie: Honoraria. Sportoletti:AbbVie: Honoraria; Janssen: Honoraria. Barr:Morphosys: Consultancy; Gilead: Consultancy; AstraZeneca: Consultancy, Research Funding; Verastem: Consultancy; Seattle Genetics: Consultancy; TG therapeutics: Consultancy, Research Funding; Abbvie/Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy; Merck: Consultancy; Genentech: Consultancy; Janssen: Consultancy. Ruchlemer:Abbvie Inc: Consultancy, Research Funding. Kersting:Celgene: Other: travel grant; Janssen: Research Funding; Abbvie: Research Funding. Huntington:Pharmacyclics: Honoraria; AbbVie: Consultancy; Novartis: Consultancy; Genentech: Consultancy; DTRM: Research Funding; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; Astrazeneca: Honoraria; TG Therapeutics: Research Funding. Herishanu:Roche: Honoraria; Sanofi: Honoraria; Medison: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; AstraZeneca: Honoraria. Jacobs:TG Therapeutics, Inc.: Research Funding; Astra Zeneca: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Pharmacyclics: Research Funding, Speakers Bureau; Seattle Genetics: Consultancy; Verastem: Consultancy; Janssen: Consultancy, Speakers Bureau; Genentech: Speakers Bureau; Sanofi Genzyme: Speakers Bureau. Portell:BeiGene: Consultancy, Research Funding; Pharmacyclics: Consultancy; TG Therapeutics: Research Funding; Infinity: Research Funding; Roche/Genentech: Consultancy, Research Funding; Xencor: Research Funding; Bayer: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Kite: Consultancy, Research Funding; Acerta/AstraZeneca: Research Funding; AbbVie: Research Funding. Rambaldi:Sanofi: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Astellas: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); BMS/Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); University of Milan: Current Employment; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Support of parent study and funding of editorial support. Received travel support., Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support from Gilead.; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Research grant from Amgen Inc.; Omeros: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company). Advisory board and speaker fees from Pfizer.. Brander:Verastem: Consultancy, Honoraria, Other, Research Funding; NCCN: Other; Novartis: Consultancy, Other; Teva: Consultancy, Honoraria; Tolero: Research Funding; NCCN: Other; Novartis: Consultancy, Other; Teva: Consultancy, Honoraria; Tolero: Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding; ArQule: Consultancy, Other, Research Funding; Ascentage: Other, Research Funding; AstraZeneca: Consultancy, Honoraria, Other, Research Funding; BeiGene: Other, Research Funding; DTRM: Other, Research Funding; Genentech: Consultancy, Honoraria, Other, Research Funding; Juno/Celgene/BMS: Other, Research Funding; MEI Pharma: Other, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Other, Research Funding; Pfizer: Consultancy, Other; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other, Research Funding. Rossi:Abbvie: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Advisory board; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Pfizer: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Coscia:Karyopharm Therapeutics: Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Coombs:Abbvie: Consultancy, Honoraria; Genentech: Honoraria; AstraZeneca: Honoraria; MEI Pharma: Honoraria; LOXO Oncology: Honoraria; Octapharma: Honoraria; Novartis: Honoraria. Schuster:Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria. Foà:Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Incyte: Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Cuneo:Astra Zeneca: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Bosch:Jansen: Honoraria; Abbvie: Honoraria; Novartis: Honoraria; Astra Zeneca: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Roche: Honoraria. Stamatopoulos:AstraZeneca: Honoraria; Janssen, Gilead, Abbvie: Honoraria, Research Funding. Ghia:Adaptive, Dynamo: Consultancy, Honoraria; Novartis: Research Funding; BeiGene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Celgene/Juno: Consultancy, Honoraria; Lilly: Consultancy, Honoraria; MEI: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Gilead: Consultancy, Honoraria, Research Funding; ArQule: Consultancy, Honoraria; Acerta/AstraZeneca: Consultancy, Honoraria. Mato:Adaptive: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; BeiGene: Consultancy; LOXO: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; TG Therapeutics: Consultancy, Other: DSMB, Research Funding.

Here we report on novel and potent pyridyl-cycloalkyl-carboxylic acid inhibitors of microsomal prostaglandin E synthase-1 (PTGES). PTGES produces, as part of the prostaglandin pathway, prostaglandin E2 which is a well-known driver for pain and inflammation. This fact together with the observed upregulation of PTGES during inflammation suggests that blockade of the enzyme might provide a beneficial treatment option for inflammation related conditions such as endometriosis. Compound 5a, a close analogue of the screening hit, potently inhibited PTGES in vitro, displayed excellent PK properties in vitro and in vivo and demonstrated efficacy in a CFA-induced pain model in mice and in a rat dyspareunia endometriosis model and was therefore selected for further studies.

Background Age-related declines in function can limit older adults' independence with activities of daily living (ADLs). While task-specific training maybe a viable approach to improve function, limited clinical resources prevent extensive training on wide ranges of skills and contexts. Thus, training on one task for the benefit of another (i.e., transfer) is important in geriatric physical rehabilitation. The purpose of this study was to test whether motor transfer would occur between two functionally different upper extremity tasks that simulate ADLs in a sample of older adults following task-specific training. Methods Ninety community dwelling adults ages 43 to 94 years old performed two trials of a functional dexterity and functional reaching task at baseline, and were then assigned to one of two groups. The training group completed 3 days of task-specific training (150 trials) on the functional reaching task, whereas the no-training group received no training on either task. Both groups were re-tested on both tasks at the end of Day 3. Results No significant interactions were observed between group (training vs. no-training) and time (baseline vs. re-test) on the functional dexterity task (i.e. transfer task), indicating no difference in the average amount of change from baseline to re-test between the groups. However, post hoc bivariate linear regression revealed an effect of age on motor transfer within the training group. For those who trained on the functional reaching task, the amount of transfer to the dexterity task was inversely related to age. There was no significant relationship between age and motor transfer for the no-training group. Discussion and conclusions Results of our a priori group analysis suggest that functional reaching training did not, on average, transfer to the dexterity task. However, post hoc regression analysis showed that motor transfer was both experience- and age-dependent, such that motor transfer may decline with advanced age. Future research will consider how functional and cognitive aging influences transfer of motor skills across different activities of daily living.

In humans, apolipoprotein C-III (apoC-III) plasma levels have been associated with increased risk of cardiovascular disease. This association is in part explained by the effects of apoC-III on triglyceride (TG) metabolism; apoC-III raises plasma TG by increasing very low density lipoprotein (VLDL) secretion, inhibiting lipoprotein lipase (LPL)-mediated TG lipolysis, and impairing the removal of triglyceride-rich lipoprotein (TRL) remnants from the circulation. In this study, we explored the structure-function relationship the interaction of apoC-III with plasma lipoproteins and its ultimate impact on LPL activity. The structural and functional properties of wild-type (WT) apoC-III were compared with two missense variants previously associated with lower (A23T) and higher (Q38K) plasma TG. ApoC-III in the lipid-free state is unstructured but its helix content and stability increases when bound to lipid. Lipid-bound apoC-III contains two alpha helices spanning residues amino acids 11 - 38 (helix 1) and 44 – 64 (helix 2). Investigation of the structural and functional consequences of the A23T and Q38K variants showed that these amino acid substitutions within helix 1 do not significantly alter the stability of the helical structure but affect its hydrophilic-lipophilic properties. The A23T substitution impairs lipoprotein binding capacity, reduces LPL inhibition, and ultimately leads to lower plasma TG levels. Conversely, the Q to K substitution at position 38 enhances the lipid affinity of helix 1, increases TRL binding capacity and LPL inhibition, and is associated with hypertriglyceridemia. This study indicates that structural modifications that perturb the hydrophilic/lipophilic properties of the alpha helices can modulate the hypertriglyceridemic effects of apoC-III.

There is a Blood Commentary on this article in this issue., Key Points Both watch-and-wait and treated CLL patients have high mortality rates when admitted for COVID-19. Receiving a BTKi for CLL at COVID-19 diagnosis severe enough to require hospitalization did not influence case fatality rate in this study., Given advanced age, comorbidities, and immune dysfunction, chronic lymphocytic leukemia (CLL) patients may be at particularly high risk of infection and poor outcomes related to coronavirus disease 2019 (COVID-19). Robust analysis of outcomes for CLL patients, particularly examining effects of baseline characteristics and CLL-directed therapy, is critical to optimally manage CLL patients through this evolving pandemic. CLL patients diagnosed with symptomatic COVID-19 across 43 international centers (n = 198) were included. Hospital admission occurred in 90%. Median age at COVID-19 diagnosis was 70.5 years. Median Cumulative Illness Rating Scale score was 8 (range, 4-32). Thirty-nine percent were treatment naive (“watch and wait”), while 61% had received ≥1 CLL-directed therapy (median, 2; range, 1-8). Ninety patients (45%) were receiving active CLL therapy at COVID-19 diagnosis, most commonly Bruton tyrosine kinase inhibitors (BTKi’s; n = 68/90 [76%]). At a median follow-up of 16 days, the overall case fatality rate was 33%, though 25% remain admitted. Watch-and-wait and treated cohorts had similar rates of admission (89% vs 90%), intensive care unit admission (35% vs 36%), intubation (33% vs 25%), and mortality (37% vs 32%). CLL-directed treatment with BTKi’s at COVID-19 diagnosis did not impact survival (case fatality rate, 34% vs 35%), though the BTKi was held during the COVID-19 course for most patients. These data suggest that the subgroup of CLL patients admitted with COVID-19, regardless of disease phase or treatment status, are at high risk of death. Future epidemiologic studies are needed to assess severe acute respiratory syndrome coronavirus 2 infection risk, these data should be validated independently, and randomized studies of BTKi’s in COVID-19 are needed to provide definitive evidence of benefit., Visual Abstract

The superfamily of massively large AAA+ protein molecular machines functions to convert the chemical energy of cytosolic ATP into physicomechanical form and use it to perform an extraordinary number of physical operations on proteins, nucleic acids, and membrane systems. Cryo-EM studies now reveal some aspects of substrate handling at high resolution, but the broader interpretation of AAA+ functional properties is still opaque. This paper integrates recent hydrogen exchange results for the typical AAA+ protein Hsp104 with prior information on several near and distantly related others. The analysis points to a widely conserved functional strategy. Hsp104 cycles through a long-lived loosely-structured energy-input "open" state that releases spent ADP and rebinds cytosolic ATP. ATP-binding energy is transduced by allosteric structure change to poise the protein at a high energy level in a more tightly structured "closed" state. The briefly occupied energy-output closed state binds substrate strongly and is catalytically active. ATP hydrolysis permits energetically downhill structural relaxation, which is coupled to drive energy-requiring substrate processing. Other AAA+ proteins appear to cycle through states that are analogous functionally if not in structural detail. These results revise the current model for AAA+ function, explain the structural basis of single-molecule optical tweezer kinetic phases, identify the separate energetic roles of ATP binding and hydrolysis, and specify a sequence of structural and energetic events that carry AAA+ proteins unidirectionally around a functional cycle to propel their diverse physical tasks.

Background: Up to 50% of patients suffering from Non-Hodgkin`s lymphoma (NHL) become refractory to or relapse after treatment (M. Crump, Blood 2017). With this, the lack of curative outcomes for patients with both indolent and aggressive NHL subtypes remains an unmet medical need. The CD20 CD3 T cell bispecific antibody glofitamab induces specific T-cell activation and has demonstrated significant single agent activity in r/r NHL patients (NP30179 study, M. Dickinson, EHA 2020, Abstract S241). RO7227166, a CD19 targeted 4-1BBL (CD137) costimulatory agonist has shown synergistic anti-tumor activity when combined with glofitamab in preclinical models (fig 1). RO7227166 is a bispecific antibody-like fusion protein composed of a split trimeric 4-1BB ligand, a tumor antigen-targeting moiety recognizing CD19, and a silent Fc part preventing Fc-mediated toxicity. 4-1BB is an inducible co-stimulatory molecule expressed by activated T-cells or NK cells. Through CD19-binding, the 4-1BB ligand moiety can deliver co-stimulatory signals to activated T- and NK-cell subsets in the tumor. The expected mode of action (MoA) for this molecule is to deliver a costimulatory signal 2 to enhance the effector function of tumor-infiltrating T cells or NK cells upon their activation (signal 1) by a T-cell bispecific antibody (e.g. glofitamab, RO7082859) or a tumor-targeted ADCC antibody (e.g. obinutuzumab). By delivering direct T-cell-target cell engagement followed by costimulatory activation the aim is to offer a highly active off-the-shelf immunotherapy combination. Methods: RO7227166 is being developed in combination with glofitamab and obinutuzumab in a phase I, open-label, dose-escalation study BP41072 (NCT04077723). The study is designed to evaluate the combination maximum tolerated dose (MTD), safety, tolerability, pharmacokinetic (PK), and/or pharmacodynamic (PD) profile of escalating doses of RO7227166, and to evaluate preliminary anti-tumor activity in participants with r/r NHL. The dose escalation stage is divided into Part I (combination with obinutuzumab) and Part II (combination with glofitamab) followed by an expansion stage (Part III). During Part I patients receive 1000mg obinutuzumab intravenously (IV) at a q3w schedule in combination with CD19 4-1BBL IV. During part II glofitamab is given in a q3w schedule with RO7227166 introduced at C2D8 and administered concomitantly from C3D1 onwards. A fixed dose of obinutuzumab (Gpt; pre-treatment) is administered seven days prior to the first administration of RO7227166 and seven days prior to the first administration of glofitamab (M. Bacac, Clin Cancer Res 2018; M. Dickinson, EHA 2020, Abstract S241). Patients will initially be recruited into part I of the study only using single-participant cohorts, where a rule-based dose-escalation is implemented, with dosing initiated at 5 μg (flat dose). As doses of RO7227166 increase, multiple participant cohorts will be recruited and dose-escalation will be guided by the mCRM-EWOC design for overdose control. Commencement of Part II including decision on the RO7227166 starting dose will be guided by safety and PK data from Part I. Patients with r/r NHL meeting standard organ function criteria and with adequate blood counts will be eligible. The maximum duration of the study for each participant will be up to 24 months in Part I (excluding survival follow-up) and up to 18 months in Part II and Part III. Tumor biopsies and peripheral blood biomarker analyses will be used to demonstrate MoA and proof of concept of an off the shelf flexible combination option providing signals 1 and 2. Disclosures Hutchings: Takeda: Honoraria; Takeda: Research Funding; Genmab: Honoraria; Roche: Honoraria; Genmab: Research Funding; Janssen: Research Funding; Novartis: Research Funding; Sankyo: Research Funding; Roche: Consultancy; Genmab: Consultancy; Takeda: Consultancy; Roche: Research Funding; Celgene: Research Funding; Daiichi: Research Funding; Sanofi: Research Funding. Bosch:Hoffmann-La Roche: Research Funding. Gritti:Italfarmaco: Consultancy; F. Hoffmann-La Roche Ltd: Honoraria; Jannsen: Other: Travel Support; Autolus: Consultancy; IQVIA: Consultancy; Kite: Consultancy; Takeda: Honoraria; Amgen: Honoraria. Carlo-Stella:Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, AstraZeneca: Honoraria; Servier, Novartis, Genenta Science srl, ADC Therapeutics, F. Hoffmann-La Roche, Karyopharm, Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics and Rhizen Pharmaceuticals: Research Funding; Boehringer Ingelheim and Sanofi: Consultancy. Townsend:Roche, Gilead: Consultancy, Honoraria. Morschhauser:Gilead: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Servier: Consultancy; Janssen: Honoraria; Epizyme: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy. Cartron:Celgene: Consultancy, Honoraria; F. Hoffmann-La Roche: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Honoraria; Jansen: Honoraria; Gilead: Honoraria. Ghesquieres:CELGENE: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Roche: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Gilead: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES; Janssen: Honoraria. de Guibert:Gilead Sciences: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Herter:Roche Glycart AG: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Korfi:Roche Diagnostics GmbH: Consultancy. Craine:Roche: Current Employment. Mycroft:Roche: Current Employment. Whayman:Roche: Current Employment. Mueller:Roche: Current Employment. Dimier:Roche: Current Employment. Moore:Roche: Current Employment. Belli:Roche Pharma: Current Employment. Kornacker:Hoffmann-La Roche Ltd.: Current Employment, Current equity holder in publicly-traded company. Lechner:Roche Diagnostics GmbH: Current Employment, Current equity holder in publicly-traded company. Dickinson:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Merck Sharp & Dohme: Consultancy; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

SummaryThe benzdiimidazole NAB2 rescues α-synuclein-associated trafficking defects associated with early onset Parkinson’s disease in a Nedd4-dependent manner. Despite identification of E3 ubiquitin ligase Nedd4 as a putative target of NAB2, its molecular mechanism of action has not been elucidated. As such, the effect of NAB2 on Nedd4 activity and specificity was interrogated through biochemical, biophysical, and proteomic analyses. NAB2 was found to bind Nedd4 (KDapp = 42 nM), but this binding is side chain mediated and does not alter its conformation or ubiquitination kinetics in vitro. Nedd4 co-localizes with trafficking organelles, and NAB2 exposure did not alter its colocalization. Ubiquitin-enrichment coupled proteomics revealed that NAB2 stimulates ubiquitination of trafficking and transport associated proteins, most likely through modulating the substrate specificity of Nedd4, providing a putative protein network involved in the NAB2 mechanism.

Phage G has the largest capsid and genome of any known propagated phage. Many aspects of its structure, assembly, and replication have not been elucidated. Herein, we present the dsDNA-packed and empty phage G capsid at 6.1 Å and 9 Å resolution, respectively, using cryo-EM for structure determination and mass spectrometry for protein identification. The major capsid protein, gp27, is identified and found to share the HK97-fold universally conserved in all previously solved dsDNA phages. Trimers of the decoration protein, gp26, sit on the 3-fold axes and are thought to enhance the interactions of the hexameric capsomeres of gp27, for other phages encoding decoration proteins. Phage G’s decoration protein is longer than what has been reported in other phages, and we suspect the extra interaction surface area helps stabilize the capsid. We identified several additional capsid proteins, including a candidate for the prohead protease responsible for processing gp27. Furthermore, cryo-EM reveals a range of partially full, condensed DNA densities that appear to have no contact with capsid shell. Three analyses confirm that the phage G host is a Lysinibacillus, and not Bacillus megaterium: identity of host proteins in our mass spectrometry analyses, genome sequence of the phage G host and host range of phage G.

Significance Despite causing regular outbreaks with high case fatality rates, there are currently no licensed vaccines or therapeutics for Nipah virus (NiV) infection. Here, we sought to determine the molecular basis for how the antibody response neutralizes NiV by targeting the surface-displayed fusion glycoprotein, NiV-F. Our structural study reveals a neutralizing antibody epitope at the membrane-distal portion of the prefusion trimeric NiV-F, which is well conserved across known NiV strains. Further structure–function analyses demonstrate that additional antibodies bind this region of the NiV-F apex, suggesting that this is an immunologically accessible site of vulnerability. This work reveals the membrane-distal regions of NiV-F and the F glycoprotein from closely related Hendra virus as attractive targets for antiviral and vaccine development., Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes frequent outbreaks of severe neurologic and respiratory disease in humans with high case fatality rates. The 2 glycoproteins displayed on the surface of the virus, NiV-G and NiV-F, mediate host-cell attachment and membrane fusion, respectively, and are targets of the host antibody response. Here, we provide a molecular basis for neutralization of NiV through antibody-mediated targeting of NiV-F. Structural characterization of a neutralizing antibody (nAb) in complex with trimeric prefusion NiV-F reveals an epitope at the membrane-distal domain III (DIII) of the molecule, a region that undergoes substantial refolding during host-cell entry. The epitope of this monoclonal antibody (mAb66) is primarily protein-specific and we observe that glycosylation at the periphery of the interface likely does not inhibit mAb66 binding to NiV-F. Further characterization reveals that a Hendra virus-F–specific nAb (mAb36) and many antibodies in an antihenipavirus-F polyclonal antibody mixture (pAb835) also target this region of the molecule. Integrated with previously reported paramyxovirus F−nAb structures, these data support a model whereby the membrane-distal region of the F protein is targeted by the antibody-mediated immune response across henipaviruses. Notably, our domain-specific sequence analysis reveals no evidence of selective pressure at this region of the molecule, suggestive that functional constraints prevent immune-driven sequence variation. Combined, our data reveal the membrane-distal region of NiV-F as a site of vulnerability on the NiV surface.

Hsp104 is a hexameric AAA(+) ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5′-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo. We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide–binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104–RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

This single-arm, open-label, phase 1b study evaluated the maximum tolerated dose (MTD) of venetoclax when given with obinutuzumab and its safety and tolerability in patients with relapsed/refractory (R/R) or previously untreated (first line [1L]) chronic lymphocytic leukemia (CLL). Venetoclax dose initially was escalated (100-400 mg) in a 3 + 3 design to define MTD combined with standard-dose obinutuzumab. Patients received venetoclax (schedule A) or obinutuzumab (schedule B) first to compare safety and determine dose/schedule for expansion. Venetoclax-obinutuzumab was administered for 6 cycles, followed by venetoclax monotherapy until disease progression (R/R) or fixed duration 1-year treatment (1L). Fifty R/R and 32 1L patients were enrolled. No dose-limiting toxicities were observed. Safety, including incidence of tumor lysis syndrome (TLS), did not differ between schedules (2 laboratory TLSs per schedule). Schedule B and a 400-mg dose of venetoclax were chosen for expansion. The most common grade 3-4 adverse event was neutropenia (R/R, 58% of patients; 1L, 53%). Rates of grade 3-4 infections were 29% (R/R) and 13% (1L); no fatal infections occurred in 1L. All infusion-related reactions were grade 1-2, except for 2 grade 3 events. No clinical TLS was observed. Overall best response rate was 95% in R/R (complete response [CR]/CR with incomplete marrow recovery [CRi], 37%) and 100% in 1L (CR/CRi, 78%) patients. Rate of undetectable (

OBJECTIVE:To investigate the impact of metabolic syndrome and its components on osteoarthritis of the hip joints compared to a healthy cohort in the KORA MRI-study. METHODS:Randomly selected men and women from the general population were classified as having metabolic syndrome, defined as presence of central obesity plus two of the following four components: elevated blood pressure (BP), elevated fasting glucose, elevated triglycerides (TG) and low HDL-cholesterol (HDL-c), or as controls without metabolic syndrome. Therefore, each subject underwent detailed assessment of waist circumference as well as fasting glucose, systolic and diastolic BP, TG, and HDL-c concentrations as well as a full-body MR scan. MR measurements were performed on a 3 Tesla scanner (Magnetom Skyra, Siemens) including a dual-echo Dixon and a T2 SS-FSE sequence for anatomical structures. In order to quantify osteoarthritis of the hip, assessment was performed by two independent, experienced radiologists for joint gap narrowing, osteophytic lipping and subchondral changes (e.g. sclerosis, pseudocysts). Associations between metabolic syndrome components and hip degeneration were estimated by logistic regression models providing odds ratios. RESULTS:Among 354 included participants (mean age: 56.1 ± 9.2 years; 55.4% male), 119 (34%) had metabolic syndrome, while 235 (66%) were part of the control group. Except for elevated blood glucose (p = 0.02), none of the metabolic syndromes' component was independently associated with osteoarthritis. Multivariable adjusted ORs for osteoarthritis of the right hip were 1.00 (95% CI 0.98;1.03), 1.00 (95% CI 0.99;1.00), 1.01 (95% CI 0.99;1.03), 1.00 (95% CI 0.97;1.04) and 1.01 (95% CI 0.96;1.06), and for the left hip 1.00 (95% CI 0.98;1.03), 1.00 (95% CI 1.00;1.01), 1.01 (95% CI 0.99;1.03), 0.99 (95% CI 0.96;1.02) and 1.04 (95% CI 0.99;1.09) for waist circumference, triglyceride, HDL-c and systolic and diastolic BP, respectively. Blood glucose was a borderline non-dependent factor for osteoarthritis of the right hip (OR: 1.02 (95% CI 1.0;1.04); p = 0.05). Furthermore, the compound metabolic syndrome was not significantly associated (OR left hip: 1.53 (95% CI 0.8;2.92), p = 0.20; OR right hip: 1.33 (95% CI 0.72;2.45), p = 0.37) with osteoarthritis of the hip joint. Age as well as gender (left hip) were the only parameters in univariate and multivariate analysis to be significantly associated with osteoarthritis of the hip joint. CONCLUSION:The compound metabolic syndrome showed no association with osteoarthritis of the hip joint. Age was the only parameter to be dependently and independently associated to osteoarthritis of both hip joints, while elevated blood glucose was independently associated with degeneration of the right hip joint.

Advanced hydrogen exchange (HX) methodology can now determine the structure of protein folding intermediates and their progression in folding pathways. Key developments over time include the HX pulse labeling method with nuclear magnetic resonance analysis, the fragment separation method, the addition to it of mass spectrometric (MS) analysis, and recent improvements in the HX MS technique and data analysis. Also, the discovery of protein foldons and their role supplies an essential interpretive link. Recent work using HX pulse labeling with MS analysis finds that a number of proteins fold by stepping through a reproducible sequence of native-like intermediates in an ordered pathway. The stepwise nature of the pathway is dictated by the cooperative foldon unit construction of the protein. The pathway order is determined by a sequential stabilization principle; prior native-like structure guides the formation of adjacent native-like structure. This view does not match the funneled energy landscape paradigm of a very large number of folding tracks, which was framed before foldons were known and is more appropriate for the unguided residue-level search to surmount an initial kinetic barrier rather than for the overall unfolded-state to native-state folding pathway.

Many older adults report difficulty performing one or more activities of daily living. These difficulties may be attributed to cognitive decline and as a result, measuring cognitive status among aging adults may help provide an understanding of current functional status. The purpose of the present investigation was to determine the association between cognitive status and measures of physical functioning. Seventy-six older adults participated in this study; 41 were categorized as normal memory function (NM) and 35 were poor memory function (PM). NM participants had significantly higher physical function as measured by Short Physical Performance Battery (SPPB; 9.4 ± 2.2 vs. 8.4 ± 2.0; p = .03) and peak velocity (0.67 ± 0.16 vs. 0.56 ± 0.19; p = .04) during a quick sit-to-stand task. Dual-task walking velocities were 22% and 126% slower between cognitive groups for the fast and habitual trials, respectively when compared to the single-task walking condition. Significant correlations existed between measures of memory and physical function. The largest correlations with memory were for peak (r = 0.42) and average (r = 0.38) velocity. The results suggest a positive relationship between physical function and cognitive status. However, further research is needed to determine the mechanism of the underlying relationships between physical and cognitive function.

Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8+ T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo., Human leucocyte antigen E (HLA-E) directly engages NK cells but also presents antigen to CD8+ T cells. Here the authors show crystal structures of HLA-E in complex with peptides derived from HIV and Mycobacterium tuberculosis, and describe binding conformations, the positional impact of residues involved and discuss implications for functional presentation to CD8+ T cells.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. We report a method for serial X-ray crystallography at X-ray free-electron lasers (XFELs), which allows for full use of the current 120-Hz repetition rate of the Linear Coherent Light Source (LCLS). Using a micropatterned silicon chip in combination with the high-speed Roadrunner goniometer for sample delivery, we were able to determine the crystal structures of the picornavirus bovine enterovirus 2 (BEV2) and the cytoplasmic polyhedrosis virus type 18 polyhedrin, with total data collection times of less than 14 and 10 min, respectively. Our method requires only micrograms of sample and should therefore broaden the applicability of serial femtosecond crystallography to challenging projects for which only limited sample amounts are available. By synchronizing the sample exchange to the XFEL repetition rate, our method allows for most efficient use of the limited beam time available at XFELs and should enable a substantial increase in sample throughput at these facilities.

To the editor: The inhibitor of Bruton’s tyrosine kinase (BTK) ibrutinib has transformed the treatment of chronic lymphocytic leukemia (CLL); many patients with previously untreatable disease may now enter durable remissions.[1][1],[2][2] Nevertheless, the kinome of ibrutinib is broad, resulting

Background CCS1477 is a first in class potent, selective and orally bioavailable inhibitor of the bromodomains of p300 and CBP, two closely related histone acetyl transferases with oncogenic roles in haematological malignancies. In pre-clinical studies, CCS1477 was found to be a potent inhibitor of cell proliferation in acute myeloid leukaemia (AML) multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) cell lines. In primary patient AML blast cells CCS1477 inhibited proliferation through a combination of cell cycle arrest at the G1/S transition and an induction of differentiation (up-regulation of CD11b and CD86). CCS1477 has significant anti-tumour activity, inducing tumour regressions in xenograft models of AML and MM. These effects were accompanied by significant reductions in tumour MYC and IRF4 expression. Additionally, there are molecular features of certain haematological malignancies that are likely to increase the sensitivity to p300/CBP inhibition with CCS1477. For example, in B-cell lymphomas there are frequent loss of function mutations in CBP that are associated with heightened sensitivity to pre-clinical inhibition of corresponding non-mutated p300. CCS1477 represents a novel and differentiated approach to inhibiting cell proliferation and survival and offers a potential new therapeutic option for patients who have relapsed or are refractory to current standard of care therapies in AML, MM or NHL. Study Design and Methods This study is the first time that CCS1477 is being dosed in patients with haematological malignancies. The Phase I/IIa study aims to determine the maximum tolerated dose (MTD) and/or recommended Phase II dose (RP2D) and schedule(s) of CCS1477 and investigate clinical activity of CCS1477 monotherapy in patients with haematological malignancies. This study will also characterise the pharmacokinetics (PK) of CCS1477 and explore potential biological activity by assessing pharmacodynamic and exploratory biomarkers. The trial aims to enrol approximately 90 patients and is currently recruiting in the UK with plans to open additional sites in the USA. Key inclusion criteria include patients with confirmed (per standard disease specific diagnostic criteria), relapsed or refractory haematological malignancies (AML, MM and NHL). Patients must have received standard therapy which for the majority of therapeutic indications is at least 2 prior lines of therapy. Single dose and steady state pharmacokinetics will be determined in all patients. AML response will be measured in bone marrow samples. Myeloma response will be evaluated according to the 'International Myeloma Working Group Response Criteria' based on changes in M protein in blood and/or urine, changes in serum free light chains if measurable, and changes on imaging and/or bone marrow if applicable and according to the guidelines. In NHL patients, tumour assessments will be done for measurable disease, non-measurable disease, and new lesions on CT (or magnetic resonance imaging [MRI]) and/or combined with visual assessment of [18F]2-fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) for response assessment per recent International Working Group consensus criteria (RECIL 2017), until progression The study will begin with two parallel monotherapy dose-escalation arms; Arm 1: Relapsed or refractory NHL and MM; Arm2: Relapsed or refractory AML/high-risk MDS. Once a recommended phase 2 dose/schedule is reached, three monotherapy expansion arms will be opened in AML/high-risk MDS (15 patients), MM (15 patients) and NHL (30 patients). Blood samples along with bone marrow biopsies and aspirates will be collected for exploratory biomarker analysis to understand mechanisms of response to treatment or disease progression. This will include the analysis of tumour-specific and circulating biomarkers, such as tumour DNA, mRNA, proteins or metabolites. In NHL patients, analysis of CBP (and p300) mutations will be undertaken to allow retrospective correlation with tumour response and to determine if loss of function mutations in the genes for either proteins can be utilised as response predictive biomarkers in future studies. Disclosures Clegg: CellCentric Ltd: Employment, Equity Ownership. Brooks:CellCentric Ltd: Employment, Equity Ownership. Ashby:CellCentric Ltd: Employment, Equity Ownership. Pegg:CellCentric Ltd: Employment, Equity Ownership. West:CellCentric Ltd: Employment, Equity Ownership. Somervaille:Novartis: Consultancy. Knapper:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Davies:ADCT Therapeutics: Honoraria, Research Funding; MorphoSys AG: Honoraria, Membership on an entity's Board of Directors or advisory committees; BioInvent: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Karyopharma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Honoraria, Research Funding; GSK: Research Funding; Pfizer: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Introduction: Prior to effective novel agent (NA) approval for CLL, alloHSCT was recommended for CLL patients (pts) with early relapse/refractory disease after purine-analogs or deletion 17p (del17p) or TP53 mutation (TP53mut) based on expert consensus and retrospectively demonstrated overall survival (OS) advantage. Since 2014, approvals of ibrutinib (ibr), venetoclax (ven), and PI3K inhibitors (PI3Ki) have led to fewer alloHSCT for CLL. While NAs are indisputably effective, many pts will eventually progress through all available NAs. In the absence of data-driven consensus regarding role of alloHSCT for CLL, decision about proceeding to transplant is currently based on disease and transplant risk, response to NAs, and pt preference. This study of CLL pts who underwent alloHSCT following NA therapy (tx) aimed to help define the role of this potentially curative modality in the era of NAs. Methods: This multicenter, retrospective cohort study examined CLL pts who underwent alloHSCT following treatment with ≥ 1 NA, including baseline clinical, prognostic, and transplant characteristics, tx preceding alloHSCT, transplant outcomes, and tx following alloHSCT. Complex karyotype (CK) and CLL status [complete remission (CR), partial remission (PR), stable disease (SD), and progression of disease (POD)] were defined per iwCLL criteria (Hallek, et al. Blood 2018). Univariate analyses utilizing COX regression evaluated association between pre-alloHSCT factors and progression free survival (PFS). PFS, OS, and non-relapse mortality (NRM) were estimated using Kaplan Meier and life table methods. Other statistics were descriptive. Results: 69 pts with CLL underwent alloHSCT following ≥ 1 NA across 14 US and EU centers, including 6 pts with Richter's transformation (RT) prior to alloHSCT. Table 1 describes baseline characteristics. Prior to alloHSCT, 78% received ibr (n=53), 39% ven (n=25), 20% PI3Ki (n=13), and 36% ≥ 2 NAs. 90% (n=62) received a NA immediately preceding alloHSCT [n=32 ibr (16% CR, 75% PR, 9% SD/POD), n=25 ven (52% CR, 40% PR, 8% SD/POD), 4 PI3Ki (75% PR, 25% SD/POD), 1 IMiD]. With a median (med) follow up 28 months (mo; range 1.2 -85), med PFS and OS from alloHSCT for the entire cohort were not reached (Figure 1A, B). PFS and OS for pts with CLL (excluding RT pts) from alloHSCT were 60% and 82% at 24 mo respectively. Poor risk disease characteristics (TP53mut, del17p, CK), prior NA exposure (ibr, ven, PI3Ki, ≥2 NAs), and transplant characteristics (matched (8/8) vs. mismatched ( To guide decision making about timing of alloHSCT, we examined pts who received 1 (n=44) vs. ≥2 (n=25) NAs. These groups were similar in terms of poor risk features (del 17p 48% vs. 38%, TP53mut 44% vs. 40%, del11q 21% vs. 36%, CK 41% vs. 54%), transplant risk (med age 60 for both groups, med HSCT-CI 0 vs. 1, matched donor 86% vs. 71%), and disease status prior to alloHSCT (CR 25% vs. 40%, PR 66% vs. 48%, SD/POD 9% vs. 12%). PFS was similar for those exposed to 1 vs. ≥ 2 NAs (Figure 1C). Disease status at time of alloHSCT and hematopoietic cell transplantation-specific comorbidity index (HCT-CI) significantly impacted PFS (Figure 1D, E). Conclusions: In the largest series of alloHSCT following NAs, data demonstrate that alloHSCT remains a viable curative strategy that can overcome adverse CLL characteristics including TP53 disruption and CK. As many pts treated with ibr and/or ven will progress or be intolerant, alloHSCT should be included in treatment algorithms for appropriate candidates. These data suggest that exposure to 1 vs. ≥2 prior NAs did not impact outcomes, though disease status at time of alloHSCT and HCT-CI are important predictors of PFS. Therefore, decision about proceeding to alloHSCT should consider comorbidities and current depth of response, as well as anticipated depth of response with the therapeutic options remaining. These data may significantly add to development of evidence-based guidelines for alloHSCT in the era of NAs. Figure 1 Disclosures Roeker: AbbVie: Equity Ownership; Abbott Laboratories: Equity Ownership. Brown:TG Therapeutics: Consultancy; Verastem: Consultancy, Research Funding; Sun Pharmaceuticals: Research Funding; Janssen: Honoraria; Teva: Honoraria; Morphosys: Other: Data safety monitoring board; Invectys: Other: Data safety monitoring board; Octapharma: Consultancy; Dynamo Therapeutics: Consultancy; Sunesis: Consultancy; Juno/Celgene: Consultancy; Genentech/Roche: Consultancy; Gilead: Consultancy, Research Funding; Catapult Therapeutics: Consultancy; AbbVie: Consultancy; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; BeiGene: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; Loxo: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Pharmacyclics: Consultancy. Dreger:AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Neovii, Riemser: Research Funding. Eyre:Roche: Honoraria; Abbvie: Honoraria, Other: Travel to Conferences; Takeda: Other: Travel to Conferences ; Gilead: Consultancy, Other: Research support, Speakers Bureau; Janssen: Honoraria, Other: Travel to Conferences . Brander:AbbVie: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria; Tolero: Research Funding; Acerta: Research Funding; TG Therapeutics: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Research Funding; Novartis: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy; BeiGene: Research Funding; DTRM Biopharma: Research Funding; MEI: Research Funding. Skarbnik:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Verastem Oncology: Honoraria, Research Funding, Speakers Bureau; Kite Pharma: Honoraria, Speakers Bureau; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Acerta: Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Honoraria, Speakers Bureau; CLL Society: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Speakers Bureau; Novartis: Speakers Bureau. Coombs:H3 Biomedicine: Research Funding. Orchard:Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Unrestricted educational award for regional meetings ; Incyte: Other: Unrestricted educational award for regional meetings ; Adienne: Other: Unrestricted educational award for regional meetings . Sauter:Juno Therapeutics: Consultancy, Research Funding; Sanofi-Genzyme: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Kite/Gilead: Consultancy; Precision Biosciences: Consultancy; Genmab: Consultancy; GSK: Consultancy. Giralt:Johnson & Johnson: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Kite: Consultancy; Amgen: Consultancy, Research Funding; Actinium: Consultancy, Research Funding; Novartis: Consultancy; Miltenyi: Research Funding; Takeda: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum Pharmaceuticals: Consultancy. Perales:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Omeros: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria; Medigene: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Kyte/Gilead: Research Funding; Nektar Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Research Funding; NexImmune: Membership on an entity's Board of Directors or advisory committees; MolMed: Membership on an entity's Board of Directors or advisory committees. Mato:Celgene: Consultancy; AbbVie: Consultancy, Research Funding; TG Therapeutics: Consultancy, Other: DSMB member , Research Funding; LOXO: Consultancy, Research Funding; DTRM Biopharma: Research Funding; Genentech: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Gilead: Research Funding; Acerta: Consultancy; Janssen: Consultancy; AstraZeneca: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Johnson & Johnson: Consultancy, Research Funding.

Elucidation of apoA-I secondary structure in spherical plasma HDL particles is essential for understanding HDL structure and function at the molecular level. To provide this information, we have applied hydrogen exchange (HX) and mass spectrometry methods to compare apoA-I secondary structure in discoidal (two apoA-I molecules/particle) and spherical (five apoA-I molecules/particle) HDL particles. The HX kinetics indicate that the locations of helical segments within the apoA-I molecules are the same in both discoidal and spherical HDL particles (approximately 10 nm hydrodynamic diameter). Helix stabilities in both types of particles are 3-5 kcal/mol, consistent with the apoA-I molecules being in a highly dynamic state with helical segments unfolding and refolding in seconds. For the spherical HDL, apoA-I fragments corresponding to residues 115-158 exhibit bimodal HX kinetics consistent with this segment adopting an inter-converting (on the timescale of tens of minutes) helix-loop configuration. The segment adopting this configuration in the 10 nm disc is shorter because the surface area available to each apoA-I molecule is apparently larger. Loop formation in the central region of the apoA-I molecule contributes to the ability of the protein to adapt to changes in available space on the HDL particle surface. Overall, apoA-I secondary structure is largely unaffected by a change in HDL particle shape from disc to sphere.

The interaction between the TNF-family molecule receptor activator of NF-kappaB ligand (RANKL) and its receptor RANK induces osteoclast formation, activation and survival in the process of bone remodelling. RANKL-RANK also plays critical roles in T-cell/dendritic cell communication and lymph-node formation and in a variety of pathologic conditions such as tumour-cell migration and bone metastasis. Both the ectodomain of mouse RANKL and the extracellular domain of mouse RANK have been cloned, expressed and purified. Crystals of RANK alone and of RANK in complex with RANKL have been obtained that are suitable for structure determination.

Summary Crystallization remains a critical step in X-ray structure determination. Because it is not generally possible to rationally predict crystallization conditions, commercial screens have been developed which sample a wide range of crystallization space. While this approach has proved successful in many cases, a significant number of proteins fail to crystallize despite being soluble and monodispersed. It is established that chemical modification can facilitate the crystallization of otherwise intractable proteins. Here we describe a method for the reductive methylation of lysine residues which is simple, inexpensive, and efficient, and report on its application to ten proteins. We describe the effect of methylation on the physico-chemical properties of these proteins, and show that it led to diffraction-quality crystals from four proteins and structures for three that had hitherto proved refractory to crystallization. The method is suited to both low- and high-throughput laboratories.

Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining `low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.

To investigate the determinants of protein hydrogen exchange (HX), HX rates of most of the backbone amide hydrogens of Staphylococcal nuclease were measured by NMR methods. A modified analysis was used to improve accuracy for the faster hydrogens. HX rates of both near surface and well buried hydrogens are spread over more than 7 orders of magnitude. These results were compared with previous hypotheses for HX rate determination. Contrary to a common assumption, proximity to the surface of the native protein does not usually produce fast exchange. The slow HX rates for unprotected surface hydrogens are not well explained by local electrostatic field. The ability of buried hydrogens to exchange is not explained by a solvent penetration mechanism. The exchange rates of structurally protected hydrogens are not well predicted by algorithms that depend only on local interactions or only on transient unfolding reactions. These observations identify some of the present difficulties of HX rate prediction and suggest the need for returning to a detailed hydrogen by hydrogen analysis to examine the bases of structure-rate relationships, as described in the companion paper (Skinner et al., Protein Sci 2012;21:996-1005).

To examine the relationship between protein structural dynamics and measurable hydrogen exchange (HX) data, the detailed exchange behavior of most of the backbone amide hydrogens of Staphylococcal nuclease was compared with that of their neighbors, with their structural environment, and with other information. Results show that H-bonded hydrogens are protected from exchange, with HX rate effectively zero, even when they are directly adjacent to solvent. The transition to exchange competence requires a dynamic structural excursion that removes H-bond protection and allows exposure to solvent HX catalyst. The detailed data often make clear the nature of the dynamic excursion required. These range from whole molecule unfolding, through smaller cooperative unfolding reactions of secondary structural elements, and down to local fluctuations that involve as little as a single peptide group or side chain or water molecule. The particular motion that dominates the exchange of any hydrogen is the one that allows the fastest HX rate. The motion and the rate it produces are determined by surrounding structure and not by nearness to solvent or the strength of the protecting H-bond itself or its acceptor type (main chain, side chain, structurally bound water). Many of these motions occur over time scales that are appropriate for biochemical function.

Release of interleukin (IL)-1β from immunocompetent cells requires formation of the NACHT, LLR and PYD domains-containing protein 3 (NLRP3) inflammasome and caspase 1 activation. Adenosine 5'-triphosphate (ATP), acting on the P2X(7) receptor, is one factor that stimulates inflammasome assembly. We show that a novel specific P2X(7) receptor antagonist, GSK1370319A, inhibits ATP-induced increase in IL-1β release and caspase 1 activation in lipopolysaccharide (LPS)-primed mixed glia by blocking assembly of the inflammasome in a pannexin 1-dependent manner. GSK1370319A also inhibits ATP-induced subregion-specific neuronal loss in hippocampal organotypic slice cultures, which is dependent on its ability to prevent inflammasome assembly in glia. Significantly, GSK1370319A attenuates age-related deficits in long-term potentiation (LTP) and inhibits the accompanying age-related caspase 1 activity. We conclude that inhibiting P2X(7) receptor-activated NLRP3 inflammasome formation and the consequent IL-1β release from glia preserve neuronal viability and synaptic activity.